Production of rabbit polyclonal antibody against apobec-1 by genetic immunization.
Circulating apolipoprotein B (apoB) exists in two forms; apoB-100 and apoB-48. ApoB-48 is a truncated form of apoB resulting from RNA editing. The editing enzyme, called apobec-1, converts a cytidine (C) at nucleotide 6666 in apoB 100 mRNA to a uridine (U) and changes a CAA codon to an in-frame stop codon, UAA. We have produced a specific rabbit polyclonal antiserum against apobec-1 by genetic immunization. The cDNA of mouse apobec-1 was inserted downstream and in-frame at the BamH I site in the last exon of human growth hormone cDNA driven by a cytomegalovirus promoter. This plasmid was injected together with another plasmid expressing granulocyte macrophage colony-stimulating factor into the thigh muscles of a rabbit. The resulting antiserum demonstrated high specificity on Western blots, and inhibited the apoB mRNA editing activity of mouse liver extract in a dose-dependent manner. This report demonstrates that DNA immunization is a powerful technique that can be readily applied to other sparse or difficult-to-purify proteins in lipid metabolism.
Yeung
S C
SC
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Anderson
J
J
Kobayashi
K
K
Oka
K
K
Chan
L
L
D000071479:APOBEC-1 Deaminase; D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000494:pharmacology*; D001055:Apolipoproteins B / Q000097:blood / Q000145:classification / Q000235:genetics; D015153:Blotting, Western; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D013006:Growth Hormone / Q000235:genetics; D007114:Immunization / Q000379:methods*; D008099:Liver / Q000201:enzymology; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D017393:RNA Editing / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D014170:Transformation, Genetic; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D000906:Antibodies; D001055:Apolipoproteins B; D018076:DNA, Complementary; D000939:Epitopes; D012333:RNA, Messenger; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013006:Growth Hormone; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605090:Apobec1 protein, mouse; D003564:Cytidine Deaminase